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Jul 18, 2018 - a catalyst, the corresponding chiral alcohols (1a–3a) were obtained with a low enantiomeric excess. (4–21% ee). Whereas the reduction of 1 ...
catalysts Article

Enantioselective Bioreduction of Prochiral Pyrimidine Base Derivatives by Boni Protect Fungicide Containing Live Cells of Aureobasidium pullulans 2 , Hanna Pawluk 1 , Renata Kołodziejska 1, *, Renata Studzinska ´ 3 Aleksandra Karczmarska-Wódzka and Alina Wo´zniak 1 1

2 3

*

Department of Medical Biology and Biochemistry, Faculty of Medicine, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun, ´ Karłowicza 24, 85–092 Bydgoszcz, Poland; [email protected] (H.P.); [email protected] (A.W.) Department of Organic Chemistry, Faculty of Pharmacy, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun, ´ Jurasza 2, 85–089 Bydgoszcz, Poland; [email protected] Department of Pharmacology and Therapy, Faculty of Medicine, Collegium Medicum in Bydgoszcz, Nicolaus Copernicus University in Torun, ´ M. Curie Skłodowskiej 9, 85–094 Bydgoszcz, Poland; [email protected] Correspondence: [email protected]; Tel.: +48-52 585-37-55

Received: 22 June 2018; Accepted: 16 July 2018; Published: 18 July 2018

 

Abstract: The enzymatic enantioselective bioreduction of prochiral 1-substituted-5-methyl-3-(2-oxo-2phenylethyl)pyrimidine-2,4(1H,3H)-diones to corresponding chiral alcohols by Boni Protect fungicide containing live cells of Aureobasidium pullulans was studied. The microbe-catalyzed reduction of bulky-bulky ketones provides enantiomerically pure products (96–99% ee). In the presence of A. pullulans (Aureobasidium pullulans), one of the enantiotopic hydrides of the dihydropyridine ring coenzyme is selectively transferred to the si sides of the prochiral carbonyl group to give secondary alcohols with R configuration. The reactions were performed under various conditions in order to optimize the procedure with respect to time, solvent, and temperature. The present methodology demonstrates an alternative green way for the synthesis of chiral alcohols in a simple, economical, and eco-friendly biotransformation. Keywords: biotransformation microbe-catalyzed reduction; prochiral ketones; stereochemistry; antifungal agent; Aureobasidium pullulans

1. Introduction Biotransformation, i.e., enzyme-catalyzed reactions, have been used over millions of years in nature to carry out reactions that are complicated from a chemical point of view. Nowadays, biocatalysis has become an important method for the production of organic compounds. Biotransformations catalyzed by isolated enzymes and by whole cells of microorganisms, in terms of microorganisms growing or resting cells, are employed for the synthesis of chemicals such as pharmaceuticals, agrochemicals, and natural products [1–3]. Bioreductions of prochiral carbonyl compounds are mainly carried out using oxidoreductases, the most frequently used of which is the catalytic potential of NADH (Nicotinamide adenine dinucleotide)/NADPH (Nicotinamide adenine dinucleotide phosphate)-dependent dehydrogenases. Dehydrogenases in living organisms catalyze the reaction of the oxidation of alcohols to carbonyl compounds by mediating the transfer of a hydride ion from the cofactor on the substrate. They also have the ability to catalyze a reverse reaction in a reduction reaction. In the reduction reaction, dehydrogenases transfer the hydride ion (pro-S or pro-R) from the cofactor to one of the prochiral Catalysts 2018, 8, 120; doi:10.3390/catal8070120

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dehydrogenases transfer the hydride ion (pro-S or pro-R) from the cofactor to one of the prochiral sides or diastereomeric diastereomeric products products [1,2]. [1,2]. sides of of the the carbonyl carbonyl group group (face (face re re or or si) si) to to give give pure pure enantiomeric enantiomeric or The main limitation to the use of dehydrogenases is the necessity of employing expensive cofactors. The main limitation to the use of dehydrogenases is the necessity of employing expensive cofactors. In In contrast, contrast, biocatalysis biocatalysis in in the the presence presence of of living living organisms organisms in in the the form form of of tissue tissue cultures cultures or or whole whole microbial cells is a very promising and effective method because the dehydrogenase, cofactor, and its microbial cells is a very promising and effective method because the dehydrogenase, cofactor, and regenerating system are all located within the cell [1,4–6]. its regenerating system are all located within the cell [1,4–6]. Baker’s most popular whole-cell biocatalyst for thefor asymmetric reductionreduction of prochiral Baker’s yeast yeastisisthethe most popular whole-cell biocatalyst the asymmetric of ketones due to its unlimited availability, ease of growing, and low cost [7]. For asymmetric prochiral ketones due to its unlimited availability, ease of growing, and low cost [7]. For asymmetric bioreduction cells of of bacterial bacterial [8], [8], bioreduction of of ketones, ketones, in in addition addition to to the the popular popular Baker's Baker's yeast, yeast, most most often often the the cells fungal fungal [9–13], [9–13], and and plant plant tissue tissue [14–17] [14–17] are are used. used. The The application application of of bioreagents bioreagents in in enantioselective enantioselective enzymatic desymmetrization of prochiral ketones leads to a broad spectrum spectrum of of chiral chiral alcohols alcohols used used as intermediates intermediates in the the syntheses syntheses of of many many pharmaceuticals pharmaceuticals and and compounds compounds presenting presenting biological biological activity [6]. Not without without significance significance is is the fact that biocatalytic reactions are conducted [6]. Not conducted under under moderate conditions conditions in inaqueous aqueoussolutions solutionswithout withoutthe the use of expensive and often reagents. use of expensive and often toxictoxic reagents. For For reason, environmentally friendly, which increases scope their applications[1,2]. [1,2]. thisthis reason, theythey are are environmentally friendly, which increases thethe scope ofof their applications In In this this work, work, we we present present the the microbial microbialbiotransformation biotransformationofofthe the1-substituted-5-methyl-3-(2-oxo-21-substituted-5-methyl-3-(2-oxophenylethyl)pyrimidine-2,4(1H,3H)-dione 2-phenylethyl)pyrimidine-2,4(1H,3H)-dioneinina a reduction reduction catalyzed catalyzed by by Boni Boni Protect Protect fungicide fungicide containing live cells Boni Protect Protect is is used in orchards because cells of of Aureobasidium Aureobasidium pullulans. pullulans. Boni because the the microorganism contained in this antifungal preparation has an antagonistic activity against a number microorganism contained in this antifungal preparation has an antagonistic activity against of Penicillium expansum, Monilinia laxa,laxa, Pezicula malicorticis). It is of phytopathogenic phytopathogenicfungi fungi(Botrytis (Botrytiscinerea, cinerea, Penicillium expansum, Monilinia Pezicula malicorticis). used most often to combat white mold. The 3-substituted pyrimidine base derivatives exhibit biological It is used most often to combat white mold. The 3-substituted pyrimidine base derivatives exhibit activity, foractivity, e.g., N-3-substituted arabinofuranosyluracils and 4-thio and analogues N-3-substituted biological for e.g., N-3-substituted arabinofuranosyluracils 4-thio of analogues of N-3uridines possess hypnotic activity [18,19]. For this reason, modification these compounds in substituted uridines possess hypnotic activity [18,19]. Forthe this reason, theofmodification of these order to obtain enantiomerically pure derivatives is important from the point of view of using them compounds in order to obtain enantiomerically pure derivatives is important from the point of view as of drugs. using them as drugs. 2. 2. Results Results and and Discussion Discussion Enantioselectivity Enantioselectivity and and the the efficiency efficiency of of the the microbial microbial catalysis catalysis are are mainly mainly determined determined by by the the steric requirements of the substrate. For instance, Baker’s yeast does not tolerate long-chain dialkyl steric requirements of the substrate. For instance, Baker’s yeast does not tolerate long-chain dialkyl ketones; ketones; however, however, one one long long alkyl alkyl chain chain is is accepted accepted if if the the methyl methyl group group has has the the other other moiety. moiety. Highly Highly stereoselective catalysis is achieved for the substrate with substituents of significantly different stereoselective catalysis is achieved for the substrate with substituents of significantly different sizes sizes [20]. [20]. The The aim aim of of the the study study was was to to use use A. A. pullulans pullulans (Aureobasidium (Aureobasidium pullulans) pullulans) to to reduce reduce the the phenacyl phenacyl of of pyrimidine base derivatives with a prochiral carbon atom where there are two bulky substituents in pyrimidine base derivatives with a prochiral carbon atom where there are two bulky substituents in the For this this reason, the vicinity vicinity (Scheme (Scheme 1). 1). For reason, they they belong belong to to the the group group of of carbonyl carbonyl compounds compounds that that are are difficult to reduce by microbiological methods. difficult to reduce by microbiological methods. O

O

N

O HO

Aureobasidium pullulans Boni Protect

N N

N

R

O

1 2 3

R = CH2CH=CH2 R = CH2CH3 R = CH3

R 1a 2a 3a

O R = CH2CH=CH2 R = CH2CH3 R = CH3

Scheme 1.1.Bioreduction Bioreduction phenacyl of pyrimidine base derivatives by Aureobasidium Scheme of of thethe phenacyl of pyrimidine base derivatives (1–3) by (1–3) Aureobasidium pullulans. pullulans.

A. pullulans was earlier successfully employed as a catalyst in the bioreduction of ethyl A. pullulans was earlier successfully employed as a catalyst in the bioreduction of ethyl 2-oxo-22-oxo-2-(10 ,20 ,30 ,40 -tetrahydro-10 ,10 ,40 ,40 -tetramethyl-60 -naphthalenyl)acetate and its amide [21], ethyl (1′,2′,3′,4′-tetrahydro-1′,1′,4′,4′-tetramethyl-6′-naphthalenyl)acetate and its amide [21], ethyl 4-chloro4-chloro-3-oxobutanoate [22,23] and ethyl 2-methyl-3-oxobutanoate [24]. In our previous work, we 3-oxobutanoate [22,23] and ethyl 2-methyl-3-oxobutanoate [24]. In our previous work, we also also presented the selective biotransformation of unsymmetrical ketones and α,β-ketoesters in the presented the selective biotransformation of unsymmetrical ketones and α,β-ketoesters in the A.

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A. pullulans catalyzed reduction. The use of Boni Protect containing A. pullulans live cells resulted in alcohols and hydroxyesters with high stereoselectivity [25,26]. Compounds 1–3 have previously been reduced by the chiral reagents commonly used in asymmetric organic synthesis. In the presence of borane/oxazaborolidine, which was generated in situ from methoxyborane and (1S, 3S, 4R, 6R)-4-amino-3,7,7-trimethylbicyclo[4.1.0]heptan-3-ol, as a catalyst, the corresponding chiral alcohols (1a–3a) were obtained with a low enantiomeric excess (4–21% ee). Whereas the reduction of 1 and 3 with (S)-2-methyl-CBS-oxazaborolidine (CBS = Corey, Bakshi, and Shibata) gives 1a and 3a with 100% yield and higher enantiomeric excess (73–85% ee) [27]. We wanted to find out if the use of A. pullulans will enable the reduction of 1–3 and whether selectivity of bioreduction can be improved as a result. The biotransformation a reaction catalyzed by A. pullulans was carried out in aqueous solution (phosphate buffer pH = 7.0) at 30 ◦ C, in the presence of glucose as the energy source and with the addition of ethanol. Under these conditions, regeneration of the cofactor takes place in situ. First, compound 1 as a model substrate was subjected to bioreduction and the reaction was completed after 3, 5, and 6 days (Table 1, entry 1–3). We found that the process of biotransformation of the compound is very slow and in order to achieve an effective performance of greater than 50%, the reaction should be carried out for at least 144 h. The enantiomerically pure product of R configuration ((R)-10 ) was obtained, regardless of at which stage the reaction was finished. Table 1. Reduction of 1 by Aureobasidium pullulans. System: Phosphate Buffer pH = 7.0, glucose pH = 7.0, glucose pH = 7.0, glucose pH = 7.0:hexane 1:1 (v/v), glucose pH = 7.0:hexane 4:1 (v/v), glucose pH = 7.0:hexane 88:2 (v/v), glucose pH = 7.0:TBME 88:2 (v/v), glucose pH = 7.0:acetonitrile 88:2 (v/v), glucose pH = 7.0:THF 88:2 (v/v), glucose pH = 7.0:propan-2-ol 88:2 (v/v), glucose pH = 7.0:[BMIM][PF6 ] 88:2 (v/v), glucose pH = 7.0:[BMIM][BF4 ] 88:2 (v/v), glucose pH = 6.5, glucose pH = 7.0, sucrose pH = 6.5, sucrose

T [h]

1 [%] a

1a [%] a

ee [%] a

72 120 144 144 144 144 144 144 144 144 144 144 144 144 144

64.6 51.8 39.6 100 95.9 90.0 96.9 98.4 98.8 93.3 90.3 88.4 33.3 24.4 57.3

35.4 48.2 60.4 4.1 10.0 3.1 1.6 1.2 6.7 9.7 11.6 66.7 75.6 42.7

99 99 99 94 99 99 99 99 99 99 99 99 99 99

a

The ee (R[%] − S[%]/R[%] + S[%]) and yield were determined by HPLC. TBME = tert-butyl methyl ether; THF = tetrahydrofuran; [BMIM] = butyl-3-methylimidazolium [PF6 ] = hexafluorophosphate; [BF4 ] = tetrafluoroborate.

In order to improve the reaction yield, it was decided to reduce 1 with the addition of organic solvents: In a two-phase system phosphate buffer (pH = 7.0): Hexane (1:1 v/v), phosphate buffer (pH = 7.0):Hexane (4:1 v/v), and with organic solvents as cosolvents (Table 1, entry 4–12). Surprisingly, regardless of the polarity of organic solvents, the performance of reduction decreased. The highest yield (11.6%) was obtained in a solution of phosphate buffer (pH = 7.0) with the addition of 2% ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4 ]). Further optimization of the reaction conditions concerned the change in pH of phosphate buffer solution and the introduction of sucrose as a carbon source in the pre-incubation stage. According to the literature, the optimal pH for the microorganism A. pullulans is slightly acidic, therefore, the reduction of 1 in an environment of pH = 6.5 was carried out [20]. The obtained results are given in Table 1 (entry 13,15). The highest yield was obtained in a solution with pH = 7.0 with sucrose (75.6%; see HPLC analysis of reduction of 1 in this condition—Figure 1).

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Figure 1. Reduction of 1 in phosphate buffer solution (pH = 7.0) with sucrose. Figure 1. Reduction of 1 in phosphate buffer solution (pH = 7.0) with sucrose.

Bioreduction in aqueous solution with A. pullulans as a catalyst was also carried out for 2 and Bioreduction in aqueous solution with A. pullulans as a catalyst was also carried out for 2 and 3. 3. For compound 2, the highest efficiency and high enantioselectivity (99% ee) were obtained in For compound 2, the highest efficiency and high enantioselectivity (99% ee) were obtained in a a phosphate buffer solution with pH = 7.0 in the presence of glucose, which was higher than the phosphate buffer solution with pH = 7.0 in the presence of glucose, which was higher than the efficiency in the solution with pH = 6.5. However, the addition of sucrose to the reaction mixture, efficiency in the solution with pH = 6.5. However, the addition of sucrose to the reaction mixture, independent of the pH of the solution, resulted in a reduction in the reaction yield (Table 2). As in independent of the pH of the solution, resulted in a reduction in the reaction yield (Table 2). As in the case of 1, the microorganism A. pullulans ensures excellent selectivity of biotransformation giving the case of 1, the microorganism A. pullulans ensures excellent selectivity of biotransformation giving chiral R-alcohol with 96–99% ee. chiral R-alcohol with 96–99% ee. Table 2. Reduction of 2 and 3 by Aureobasidium pullulans in different conditions. Table 2. Reduction of 2 and 3 by Aureobasidium pullulans in different conditions. a,b a a,b a,b a System: Phosphate BufferBuffer 2 [%] a,b ee [%]ee 3a [%] a,b [%]2a [%] a,b System: Phosphate 2 [%]2a [%]3 a[%] 3 [%] a,b 3a [%] a,bee [%] ee [%] a c c pH =pH 7.0, =glucose 7.0, glucose c pH =pH 6.5, =glucose 6.5, glucosec pH = 7.0, sucrosec c 7.0, sucrose c pH =pH 6.5, =sucrose c pH = 6.5, sucrose a

41.3 60.4 76.8 85.3

41.3 60.4 76.8 85.3

58.7 58.7 39.6 39.6 23.2 14.7 23.2

14.7

99 99 99 96

73.5 73.5 91.6 91.6 86.6 94.1 86.6

99 99 99 b96

94.1

The ee and yield were determined by HPLC; 144 h; c 30 ◦ C.

a

26.5 26.5 8.4 8.4 13.4 5.9 13.4

5.9

96 96 99 99 99 96 99

96

The ee and yield were determined by HPLC; 144 h; 30 °C. b

c

The most difficult object toto reduce was compound 3. 3. For this compound, asas forfor 2, 2, the conditions The most difficult object reduce was compound For this compound, the conditions such as phosphate buffer, pH = pH 7.0, and glucose the most After 144 h, the best144 performance such as phosphate buffer, = 7.0, and are glucose are optimal. the most optimal. After h, the best was obtained, which, however, did not exceeddid 30%. pH to 6.5 resulted performance was obtained, which, however, notInterestingly, exceed 30%. lowering Interestingly, lowering pH toin6.5 a decrease these conditions, 3a accounted for 3a only 10% of the mixture. resulted in in performance. a decrease inUnder performance. Under these conditions, accounted forreaction only 10% of the Asreaction a consequence, was obtained(R)-3a with low and with highlow enantiomeric excess (Table 2). excess mixture.(R)-3a As a consequence, was yield obtained yield and high enantiomeric Compounds 1–3 are structural analogues, differing in substituents in the N-1 position. Despite (Table 2). the similarity in the1–3 chemical structure, they arediffering reduced in in substituents the same conditions at position. differentDespite rates. Compounds are structural analogues, in the N-1 Therefore, for each of the prochiral reagents, ofthe biocatalysis should be individually the similarity in the chemical structure, theythe areconditions reduced in same conditions at different rates. optimized. on the obtained, the rate the of the reaction catalyzed by A. should pullulansbeand thus the Therefore,Based for each of results the prochiral reagents, conditions of biocatalysis individually efficiency is influenced by the sizeobtained, and chemical nature of the substituent on the nitrogenand in the optimized. Based on the results the rate of the reaction catalyzed by first A. pullulans thus pyrimidine ring. The presence of the allyl substituent increases the reaction rate. the efficiency is influenced by the size and chemical nature of the substituent on the first nitrogen in the next stage, wasallyl performed at two additional theIn pyrimidine ring.biotransformation The presence of the substituent increases the temperatures reaction rate. to improve the efficiency of the reaction: 33 ◦ C and 36 ◦ C. The rate increases with the temperatures increasing temperature, In the next stage, biotransformation wasreaction performed at two additional to improve the enzymatic is also regulated by this but only a certain temperature the efficiencycatalysis of the reaction: 33 °C and 36 dependence, °C. The reaction rate within increases with the increasing range. The optimal temperature that isischaracteristic ofby thethis enzyme ensuresbut theonly highest activity of temperature, the enzymatic catalysis also regulated dependence, within a certain the biocatalyst,range. which The results in thetemperature maximum degree conversion. of The relationship applies temperature optimal that is of characteristic theinverse enzyme ensures the highest toactivity the selectivity the process, in most casesin thethe optical purity of a product decreases along with of the of biocatalyst, which results maximum degree of conversion. The inverse anrelationship increase in temperature We expected as the increased, yield chiral applies to the[28]. selectivity of the that process, intemperature most cases the optical the purity of of a product alcohols would increase without adversely affecting theWe enantioselectivity thetemperature catalyzed reaction. decreases along with an increase in temperature [28]. expected that asofthe increased, each the twowould optimalincrease conditions in which the reductions performed at elevated theFor yield of reagent, chiral alcohols without adversely affectingwere the enantioselectivity of the temperatures were selected (Table 3). The best results were obtained for 3 at 33 ◦ C. In a phosphate catalyzed reaction.

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For each reagent, the two optimal conditions in which the reductions were performed at elevated temperatures temperatureswere wereselected selected(Table (Table3).3).The Thebest bestresults resultswere wereobtained obtainedfor for3 3atat3333°C. °C.InIna aphosphate phosphate buffer solution with pH = =7.0 with glucose (Figure 2),2),the yield ofofthe reaction increased 2-fold and buffer solution with pH 7.0 with glucose (Figure the yield the reaction increased 2-fold buffer solution with pH = 7.0 with glucose (Figure 2), the yield of the reaction increased 2-fold andand even 3-fold under the same conditions with sucrose added asasa acarbon source. even 3-fold under the same conditions with sucrose added carbon source. even 3-fold under the same conditions with sucrose added as a carbon source.

Figure 2. Reduction of 3 in phosphate buffer solution (pH = 7.0) with glucose. Figure 2. 2. Reduction ofof 3 in phosphate buffer solution (pH = 7.0) with glucose. Figure Reduction 3 in phosphate buffer solution (pH = 7.0) with glucose. Table 3. Reduction of 1–3 by Aureobasidium pullulans at 33 ◦ C and 36 ◦ C. Table 3. 3. Reduction ofof 1–3 byby Aureobasidium pullulans atat 3333 °C°C and 3636 °C. Table Reduction 1–3 Aureobasidium pullulans and °C. System

System System

T [◦ C]

1a [%] a

ee [%] a

2a [%] a

ee [%] a

3a [%] a

ee [%] a

a a ee [%] a a 2a [%] a a ee [%] a a 3a [%] a a ee [%] a a T [°C] [%] T [°C] 1a1a [%] ee [%] 2a [%] ee [%] 3a [%] ee [%] 98 98 55.755.7 99 99 98 55.7 99 99 3.7 99 3636 °C°C Nd Nd 4.44.4 9999 3.73.7 9999 Nd b Nd b ◦ b b 33 C33 °C 50.0 50.0 99 99 48.948.9 99 99 b b Nd b b Nd Phosphate buffer pH =pH 6.5,=glucose 33 °C 50.0 99 48.9 99 Nd Nd b Nd bNd Phosphate buffer glucose 36 ◦ C Phosphate buffer pH 6.5, = 6.5, glucose b b Nd b b 3636 °C°C7.0 7.07.0 96 9696 3.6 3.63.6 99 9999 Nd Nd Nd Nd Nd b b b 45.245.2 33 ◦ C33 °C 37.5 37.5 99 99 99 99 Nd bNd b b Nd Nd 33 °C 37.5 99 Nd Nd 45.2 99 Phosphate buffer pH =pH 7.0,=sucrose b Phosphate buffer sucrose 36 ◦ C36 °C 5.9 5.9 98 98 99 99 Phosphate buffer pH 7.0, = 7.0, sucrose Nd bNd b b Nd Nd b b 11.111.1 36 °C 5.9 98 Nd Nd 11.1 99 a The ee and yield were determined by HPLC; Nd b —not determined a The a b ee and yield were determined by HPLC; Nd b—not determined

33 ◦ C33 °C Nd bNd b b Nd bNd b b 64.164.1 Phosphate buffer pH = 7.0, glucose 33 °C b Nd Nd 64.1 Phosphate buffer pH = 7.0, glucose 36 ◦ C 4.4 Nd Nd b Phosphate buffer pH = 7.0, glucose b b

The ee and yield were determined by HPLC; Nd —not determined

increase was observed 2 2at °C, and over 60% yield was obtained ◦33 Aslight slight increase inyield yield was observed at33 °C, and over 60% yield was obtained inthe the AA slight increase inin yield was observed forfor 2for at 33 C, and over 60% yield was obtained inin the buffer solution with pH = 7.0 with glucose (Figure 3). buffer solution with pH = 7.0 with glucose (Figure 3). buffer solution with pH = 7.0 with glucose (Figure 3).

Figure 3. Reduction of 2 in phosphate buffer solution (pH = 7.0) with glucose. Figure 3. 3. Reduction ofof 2 in phosphate buffer solution (pH = 7.0) with glucose. Figure Reduction 2 in phosphate buffer solution (pH = 7.0) with glucose.

InIn the case ofof 1,of1, a 1, 3a ◦a3C3°C increase inin temperature resulted inin ain decrease inin performance. AA further case resulted a adecrease Inthe the case °Cincrease increase intemperature temperature resulted decrease inperformance. performance. Afurther further ◦ C adversely affected the conversion rate of each reagent, but did not increase in temperature to 36 increase increaseinintemperature temperaturetoto3636°C°Cadversely adverselyaffected affectedthe theconversion conversionrate rateofofeach eachreagent, reagent,but butdid didnot not have any practical effect on the selectivity of the process. Figure 4 shows the comparison of the yield have any practical effect on the selectivity of the process. Figure 4 shows the comparison of the yield have any practical effect on the selectivity of the process. Figure 4 shows the comparison of the yield ofof 1a–3a alcohols atat different temperatures. alcohols temperatures. of1a–3a 1a–3a alcohols atdifferent different temperatures.

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100

1a

90

2a

80

3a

Yield [%]

70 60 50 40 30 20 10 0

Figure 4. Comparison of 1a–3a 1a–3aperformance performanceatat30 30◦ °C, 33◦ C, °C,and and3636◦ C °Cinindifferent differentconditions. conditions. System Figure 4. Comparison of C, 33 System 1: 1: Phosphate buffer 7.0, glucose, 2: Phosphate buffer = 6.5,System glucose, System 3: Phosphate buffer pH =pH 7.0,=glucose, SystemSystem 2: Phosphate buffer pH = 6.5,pH glucose, 3: Phosphate Phosphate pH = 7.0, sucrose. were determined buffer pH =buffer 7.0, sucrose. Yields wereYields determined by HPLC. by HPLC.

The advantage of the microbiological method we use is a unique enantioselectivity, which The advantage of the microbiological method we use is a unique enantioselectivity, which combined with the ecological and economic aspect can be an interesting alternative to combined with the ecological and economic aspect can be an interesting alternative to organocatalysis. organocatalysis. Dehydrogenases contained in the microorganism A. pullulans selectively transfer Dehydrogenases contained in the microorganism A. pullulans selectively transfer one of the enantiotopic one of the enantiotopic hydrogen ions of the dihydropyridine ring coenzyme to the si sides of the hydrogen ions of the dihydropyridine ring coenzyme to the si sides of the prochiral carbonyl group prochiral carbonyl group and provide secondary alcohols with the R configuration. Therefore, the and provide secondary alcohols with the R configuration. Therefore, the Boni Protect could be Boni Protect could be considered as an efficient bioreagent for preparation of optically pure alcohols considered as an efficient bioreagent for preparation of optically pure alcohols of derivatives of N-1 of derivatives of N-1 substituted thymine. substituted thymine. In the case of previously reduced unsymmetrical ketones (methyl ketones or bromomethyl In the case of previously reduced unsymmetrical ketones (methyl ketones or bromomethyl ketones), in order to improve enantioselectivity, it was necessary to add appropriate inhibitors, i.e., ketones), in order to improve enantioselectivity, it was necessary to add appropriate inhibitors, i.e., additives which inhibit oxidoreductases with a specific stereopreference. Without the additives, additives which inhibit oxidoreductases with a specific stereopreference. Without the additives, reduction with A. pullulans proceeded with an enantiomeric excess ranging from 0% to 65% ee [26]. reduction with A. pullulans proceeded with an enantiomeric excess ranging from 0% to 65% ee [26]. 3. 3. Materials Materials and and Methods Methods 3.1. Analytical Methods Nuclear magnetic magneticresonance resonance (NMR) spectra performed with spectrometers Bruker spectrometers (NMR) spectra were were performed with Bruker (Billerica, (Billerica, USA,MHz). 400/700 MHz). Chemical are in reported δ ppm from tetramethylsilane MA, USA,MA, 400/700 Chemical shifts are shifts reported δ ppm in from tetramethylsilane (TMS) as (TMS) as anstandard. internal standard. an internal The enantiomeric enantiomericexcess excessofofthethe chiral products (1a–3a) determined by chiral stationary chiral products (1a–3a) was was determined by chiral stationary phase phase high-performance liquid chromatography HPLC analyses were performed on a high-performance liquid chromatography (HPLC).(HPLC). HPLC analyses were performed on a Shimadzu ® 5μ Cellulose-3, LC Column 250 × 4.6 mm, Phenomenex ® Shimadzu SCL-10A VP, column Lux SCL-10A VP, column Lux 5µ Cellulose-3, LC Column 250 × 4.6 mm, Phenomenex (Warszawa, Poland). (Warszawa, Poland). mobileand phase was n-hexane and propan-2-ol v/v) theper flow rate of The mobile phase wasThe n-hexane propan-2-ol (60:40 v/v) at the flow(60:40 rate of 0.5atmL min. and 0.5 mL per at min. at 266 nm wavelength. monitored 266and nmmonitored wavelength. The samples were incubated in an orbital shaker (VORTEMP 1550 S2050; Equimed, Cracov, Poland).

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The samples were incubated in an orbital shaker (VORTEMP 1550 S2050; Equimed, Cracov, Poland). Ketones 1–3 were obtained in our earlier work [27]. The retention times of 1–3 were 26.1 min, 24.1 min, and 27.4 min, respectively. The structure of 1a–3a was verified by 1 H NMR and spectra date was compared with the literature [27]. The absolute configurations of the chiral molecules were determined by various chiroptical methods (electronic circular dichroism (ECD) and vibrational circular dichroism (VCD)) [27]. 3.2. Reagents and Solvents The chemical substances of analytical grade were commercially available sucrose, glucose, ethyl acetate, ethanol, acetonitrile, tert-butyl methyl ether (TBME), tetrahydrofuran (THF), NaCl, MgSO4 , n-hexane for HPLC, propan-2-ol for HPLC from POCH (Polish Chemical Reagents, Gliwice, Poland), butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6 ], Fluka, Buchs, Switzerland), 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4 ], Merck, Darmstadt, Germany), and Boni Protect (Koppert Biological Systems, Wien, Austria). 3.3. General Procedure of Asymmetric Reduction by Aureobasidium pullulans For a typical experiment, to a suspension of Boni Protect (0.5 g) in 7.5 mL of potassium phosphate buffer (pH 7.0) was added 2 × 10−4 mol glucose/sucrose, and the resulting suspension was incubated in an orbital shaker (350 rpm) at specific temperature (30 ◦ C, 33 ◦ C, 36 ◦ C) for 30 min. After pre-incubation, the appropriate ketone (2.5 × 10−5 mol in 0.5 mL EtOH) was added, and stirring was continued at the same temperature. The reaction progress was monitored by Thin Layer Chromatography-TLC (the solvent system used was n-hexane:ethyl acetate 1:3 v/v). After the reaction, hyflo-super cel and ethyl acetate were added and the mixture was filtered. The celit was washed with ethyl acetate, and combined filtrates were extracted with ethyl acetate (3 × 20 mL). The organic phase was dried over anhydrous MgSO4 , and the solvent was evaporated under vacuum. After that, each reaction mixture was purified by column chromatography using n-hexane:ethyl acetate 1:3 v/v to afford the product. The enantiomeric ratios were determined on the HPLC system using a chiral column. NMR and HPLC spectra of 1–3 and 1a–3a were attached in the Supplementary Materials. 4. Conclusions In summary, we have described an eco-friendly and environmentally benign asymmetric reduction system employing easily available Boni Protect fungicide as a biocatalyst. This reduction method is simple, economical (the need of costly cofactor is eliminated), and does not require the cultivation of the bioreagent. The bioreduction of different phenacyls of pyrimidine base derivatives to corresponding optically chiral alcohols has shown an exclusively (R) configuration. Thus, this study demonstrates an inexpensive approach in the synthesis of optically pure (R)-heterocyclic compounds of biological importance. Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4344/8/7/120/s1. Figure S1: 1 H NMR spectra of 1; Figure S2: 1 H NMR spectra of 2; Figure S3: 1 H NMR spectra of 3; Figure S4: 1 H NMR spectra of 1a; Figure S5: 1 H NMR spectra of 2a; Figure S6: 1 H NMR spectra of 3a; Figure S7: 13 C NMR spectra of 1; Figure S8: 13 C NMR spectra of 2; Figure S9: 13 C NMR spectra of 3; Figure S10: 13 C NMR spectra of 1a; Figure S11: 13 C NMR spectra of 2a; Figure S12: 13 C NMR spectra of 3a; Figure S13: (S)-1a (99% ee); Figure S14: (R)-1a (96% ee); Figure S15: Reduction of 1 in phosphate buffer solution (pH = 6.5) with glucose at 30 ◦ C; Figure S16: Reduction of 1 in phosphate buffer solution (pH = 6.5) with glucose at 33 ◦ C; Figure S17: Reduction of 1 in phosphate buffer solution (pH = 7.0) with sucrose at 30 ◦ C; Figure S18: Reduction of 1 in phosphate buffer solution (pH = 7.0) with sucrose at 33 ◦ C; Figure S19: (S)-2a (99% ee); Figure S20: (R)-2a (97% ee); Figure S21: Reduction of 2 in phosphate buffer solution (pH = 7.0) with glucose at 30 ◦ C; Figure S22: Reduction of 2 in phosphate buffer solution (pH = 7.0) with glucose at 33 ◦ C; Figure S23: Reduction of 2 in phosphate buffer solution (pH = 6.5) with glucose at 30 ◦ C; Figure S24: Reduction of 2 in phosphate buffer solution (pH = 6.5) with glucose at 33 ◦ C; Figure S25: (S)-3a (99% ee); Figure S26: (R)-3a (62% ee); Figure S27: Reduction of 3 in phosphate

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buffer solution (pH = 7.0) with glucose at 30 ◦ C; Figure S28: Reduction of 3 in phosphate buffer solution (pH = 7.0) with glucose at 33 ◦ C; Figure S29: Reduction of 3 in phosphate buffer solution (pH = 7.0) with sucrose at 30 ◦ C; Figure S30: Reduction of 3 in phosphate buffer solution (pH = 7.0) with sucrose at 33 ◦ C. Author Contributions: Conceptualization, R.K.; Investigation, R.K., R.S., and A.K.-W.; Methodology, R.K.; Supervision, R.K.; Writing—original draft, R.K.; Writing—review and editing, R.S., H.P., and A.W. Funding: This research received no external funding. Acknowledgments: This work has been supported by Nicolaus Copernicus University, Collegium Medicum as part of the statutory research project in 2018, No. 275. The authors also wish to thank Bio-ferm GmbH and Technical Director PhD Christina Donat for the gift of Boni Protect. Conflicts of Interest: The authors declare no conflict of interest.

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